Hyaluronidases (HAses; E.C. 3.1.25) are a group of neutral- and acid-active enzymes found throughout the animal kingdom in diverse organisms. Hyaluronidases degrade hyaluronan (HA; also known as hyaluronic acid) and, to a lesser extent, chondroitin sulfates (for a review, see Kreil et al. 1995 Protein Sci. 4:1666-9). Vertebrate hyaluronidases are separated into two general classes: 1) the neutral hyaluronidases, such as the predominantly sperm-associated protein PH20 (Liu et al. 1996 Proc. Natl. Acad. Sci. USA 93:7832-7; Primakoff et al. 1985 J. Cell Biol. 101:2239-44; Lin et al. 1993 Proc. Natl. Acad. Sci. USA 90:10071-5); and 2) the acid-active hyaluronidases, which have a distinct pH optimum between pH 3.5 to 4.0 and have been described in extracts of liver (Fiszer-Szafarz et al. 1995 Acta Biochim Pol. 42:31-3), kidney (Komender et al. 1973 Bull. Acad. Pol. Sci. (Biol. J. 21:637-41), lung (Thet et al. 1983 Biochem. Biophys. Res. Commun. 117:71-7), brain (Margolis et al. 1972 J. Neutrochem. 19:2325-32), skin (Cashman et al. 1969 Arch. Biochem. Biophys. 135:387-95), placenta, macrophages, fibroblasts (Lien et al. 1990 Biochim Biophys. Acta 1034:318-25; Ruggiero et al. 1987 J. Dent. Res. 66:1283-7), and human plasma (De Salegui et al. 1967 Arch. Biochem. Biophys. 120:60-67).
Human urine exhibits a high specific hyaluronidase activity (approximately 100-fold that of human plasma HAse activity) (Fiszer-Szafarz et al. supra; Dicker et al. 1966 J Physiol (Lond) 186:110-120; Cobbin et al. 1962 J Physiol (Lond) 163:168-174). Urine hyaluronidase may play a role in the action of antidiuretic hormones by increasing the permeability of the nephron walls, which have an ECM rich in HA (Cobbin et al. supra; Ginetzinsky et al. 1958 Nature 182:1218-1219). However, examination of this hypothesis and further characterization of the HAse activity of urine has proved elusive, mainly because the activity is present at such low concentrations.
Hyaluronan, the main substrate for hyaluronidase, is a repeating disaccharide of [G1cNAc.beta.1-4G1cUA.beta.1-3].sub.n that exists in vivo as a high molecular weight linear polysaccharide. Degradation of hyaluronan by hyaluronidase is accomplished by either cleavage at .beta.-N-acetyl-hexosamine-[1.fwdarw.4]-glycosidic bonds or cleavage at .beta.-gluconorate-[1.fwdarw.3]-N-acetylglucosamine bonds. Hyaluronan is found in mammals predominantly in connective tissues, skin, cartilage, and in synovial fluid, and is also the main constituent of the vitreous of the eye. In connective tissue, the water of hydration associated with hyaluronan creates spaces between tissues, thus creating an environment conducive to cell movement and proliferation. Hyaluronan plays a key role in biological phenomena associated with cell motility including rapid development, regeneration, repair, embryogenesis, embryological development, wound healing, angiogenesis, and tumorigenesis (Toole 1991 Cell Biol. Extracell. Matrix, Hay (ed), Plenum Press, New York, 1384-1386; Bertrand et al. 1992 Int. J. Cancer 52:1-6; Knudson et al, 1993 FASEB J. 7:1233-1241). In addition, hyaluronan levels correlate with tumor aggressiveness (Ozello et al. 1960 Cancer Res. 20:600-604; Takeuchi et al. 1976, Cancer Res. 36:2133-2139; Kimata et al. 1983 Cancer Res. 43:1347-1354).
Hyaluronidase is useful as a therapeutic in the treatment of diseases associated with excess hyaluronan and to enhance circulation of physiological fluids and/or therapeutic agents at the site of administration. For example, hyaluronidase has been used to reduce intraocular pressure in the eyes of glaucoma patients through degradation of hyaluronan within the vitreous humor (U.S. Pat. No. 4,820,516, issued Apr. 11, 1989). Hyaluronidase has also been used in cancer therapy as a "spreading agent" to enhance the activity of chemotherapeutics and/or the accessibility of tumors to chemotherapeutics (Schuller et al., 1991, Proc. Amer. Assoc. Cancer Res. 32:173, abstract no. 1034; Czejka et al., 1990, Pharmazie 45:H.9) and has been used in combination with other chemotherapeutic agents in the treatment of a variety of cancers including urinary bladder cancer (Horn et al., 1985, J. Surg. Oncol., 28:304-307), squamous cell carcinoma (Kohno et al., 94, J. Cancer Res. Oncol., 120:293-297), breast cancer (Beckenlehner et al., 1992, J. Cancer Res. Oncol. 118:591-596), and gastrointestinal cancer (Scheithauer et al., 1988, Anticancer Res. 8:391-396). Administration of hyaluronidase also induces responsiveness of previously chemotherapy-resistant tumors of the pancreas, stomach, colon, ovaries, and breast (Baumgartner et al., 1988, Reg. Cancer Treat. 1:55-58; Zanker et al., 1986, Proc. Amer. Assoc. Cancer Res. 27:390). When added extracelluarly, hyaluronidase prevents growth of tumors transplanted into mice (De Maeyer et al., 1992, Int. J. Cancer 51:657-660), inhibits tumor formation upon exposure to carcinogens (Pawlowski et al., 1979, Int. J. Cancer 23:105-109; Haberman et al., 1981, Proceedings of the 17th Annual Meeting of the American Society of Clinical Oncology, Washington, D.C., 22:105, abstract no. 415, and is effective in the treatment of brain cancer (gliomas) when following intravenous or intramuscular injection (PCT published application no. WO88/02261, Apr. 7, 1988).
Hyaluronidase expression, and levels of hyaluron, have been associated with tumor development and progression. Levels of a secreted neutral hyaluronidase activity in carcinomas derived from ovary (Miura et al. 1995 Anal. Biochem. 225:333-40), prostate (Lokeshwar et al. 1996 Cancer Res 56:651-7), brain, melanocyte, and colon (Liu et al. 1996 Proc. Natl. Acad. Sci. USA 93:7832-7837) are higher than in normal tissue. This secreted neutral hyaluronidase activity appears similar or identical to the neutral hyaluronidase activity of the sperm hyaluronidase PH20. In contrast to neutral activity, the acid active HAse activity is significantly decreased in metastatic carcinomas of the lung, breast, and colon (Northrup et al. 1973 Clin. Biochem. 6:220-8; Kolarova et al. 1970 Neoplasma 17:641-8). Further, mice having an allele of the hyal-1 locus that is associated with lower levels of serum hyaluronidase activity exhibit faster rates of growth of transplanted tumors than mice having an hyal-1 allele that is associated with 3-fold higher hyaluronidase activity levels (Fiszer-Szafarz et al. 1989 Somat. Cell. Mol. Genet. 15:79-83; De Maeyer et al. supra).
At present, the only hyaluronidase activity available for clinical use is a hyaluronidase isolated from a testicular extract from cattle (WYDASE.RTM., Wyeth-Ayerst). The bovine extract is not optimum not only because of its non-human source, but also because the extract contains multiple types of hyaluronidases and other as yet undefined components. Recently we purified and cloned the major hyaluronidase activity of human plasma, which we termed HYAL1 (also known as hpHAse), and observed that the protein is homologous to PH-20, with 40% amino acid identity (Frost et al. 1997 Biochem Biophys Res Commun 236:10-15). Highest levels of expression of the HYAL1 mRNA were found in liver and kidney. The production of monoclonal antibodies was essential for purification of sufficient enzyme for microsequencing.
There is a clear need in the field for additional purified acid-active hyaluronidases that can be used in therapy and other applications. The present invention addresses this problem.